A nonluminescent and highly virulent Vibrio harveyi strain is associated with "bacterial white tail disease" of Litopenaeus vannamei shrimp

Abstract

Recurrent outbreaks of a disease in pond-cultured juvenile and subadult Litopenaeus vannamei shrimp in several districts in China remain an important problem in recent years. The disease was characterized by "white tail" and generally accompanied by mass mortalities. Based on data from the microscopical analyses, PCR detection and 16S rRNA sequencing, a new Vibrio harveyi strain (designated as strain HLB0905) was identified as the etiologic pathogen. The bacterial isolation and challenge tests demonstrated that the HLB0905 strain was nonluminescent but highly virulent. It could cause mass mortality in affected shrimp during a short time period with a low dose of infection. Meanwhile, the histopathological and electron microscopical analysis both showed that the HLB0905 strain could cause severe fiber cell damages and striated muscle necrosis by accumulating in the tail muscle of L. vannamei shrimp, which led the affected shrimp to exhibit white or opaque lesions in the tail. The typical sign was closely similar to that caused by infectious myonecrosis (IMN), white tail disease (WTD) or penaeid white tail disease (PWTD). To differentiate from such diseases as with a sign of "white tail" but of non-bacterial origin, the present disease was named as "bacterial white tail disease (BWTD)". Present study revealed that, just like IMN and WTD, BWTD could also cause mass mortalities in pond-cultured shrimp. These results suggested that some bacterial strains are changing themselves from secondary to primary pathogens by enhancing their virulence in current shrimp aquaculture system.

Figure 1. Gross signs of diseased L. vannamei shrimp.

(A) Gross signs of L. vannamei shrimp naturally occurred in the farm. Focal to extensive whitish muscles in the tail (black arrows) with (top) or without (bottom) red discoloration in the body and appendages (B) Gross signs of L. vannamei shrimp laboratory-infected with the V. harveyi strain HLB0905. Compared to normal shrimp (bottom, white arrow), the infected shrimp exhibited an extensive whitish or opaque appearance in the tail (top, black arrow).

Figure 2. Histopathological changes in whitish muscles.

(A and C) Normal (black arrows) and broken muscle fibers (white arrows). (B and D) Coagulative to liquefactive muscle necrosis and infiltration of a great number of rod-shaped bacteria (black arrows). Tissue A and B were sampled from L. vannamei shrimp with a WTD-like disease in the farm, while tissue C and D were sampled from L. vannamei shrimp laboratory-infected with the V. harveyi strain HLB0905(A: bar = 35 µm; B, C and D: bar = 10 µm).

Figure 3. Ultrastructural changes in whitish muscles.

(A) The mitochondrial membrane was broken (black arrow, bar = 0.5 µm). (B) The nuclear pyknosis (white arrow) and fiber cell vacuolation (black arrow, bar = 2 µm). (C) Damaged myofibrils (white arrow shows normal morphology of myofibrils) and invasion of bacteria (black arrow, bar = 0.5 µm). (D) Damaged fiber cells (white arrow shows damaged mitochondria) and infiltration of Vibrio-like bacteria (black arrow) (bar = 2 µm). All the tissues were sampled from L. vannamei shrimp with a WTD-like disease in the farm.

Figure 4. Morphology of isolated V. harveyi strain HLB0905 (bar = 2 µm).

Figure 5. PCR products amplified from the V. harveyi hemolysin gene.

These tissues were respectively sampled from four L. vannamei shrimp with a WTD-like disease in the farm. M: DL 2000 marker (Takara, Japan); 1–4, whitish muscle; 5–8, hemolymph; 9–12, hepatopancreas.

Figure 6. Phylogenetic tree based on the neighbor-joining method, using complete Vibrio hemolysin gene sequences.

Bootstrap values are expressed as percentages of 1000 replications.